KR100533244B1 - Cytotoxic Alkaloid Derivatives Including Asmarine A and B Isolated from a Sponge - Google Patents

Abstract

Ars marine A and B, the novel di-terpene cytotoxicity of the present invention the alkaloid is separated from the sponge Ras file Ria species (Raspailia sp.). The structure of the compound was established by NMR data and X-ray analysis. In the compounds having formulas (1) and (2) claimed, R ¹ is hydrogen or lower alkyl or lower alkanoyl; R ² is hydrogen or lower alkyl; R ³ is an alkyl or cycloalkyl group comprising one or more isoprene units, or one monoterpene or one sesquiterpene or diterpene group; R ⁴ and R ⁵ are hydrogen or lower alkyl; R ⁶ is lower alkyl; X is F or Cl or Br or I.

Description

Cytotoxic Alkaloid Derivatives Including Asmarine A and Ｂ Isolated from a Sponge}

The invention sponge Ras file Ria species of alkaloid, asbestos Marine (Asmarine) of novel cytotoxic separated from (Raspailia sp.) Relates to the A and B.

Marine organisms, particularly soft corals, sponges and tunicates, provide many secondary metabolites and exhibit varying degrees of biological activity (reference 1). One class of these metabolites is the diterpene-alkaloids, and in 1984 the structure of four agelasines was reported (reference 2):

We isolated a novel cytotoxic diterpene-alkaloid related to the above azelasin from the Lassilia species .

The present invention provides novel diterpene-alkaloids having the formula

In the above formula, R ¹ is hydrogen or lower alkyl or lower alkanoyl; R ² is hydrogen or lower alkyl; R ³ is an alkyl or cycloalkyl group comprising one or more isoprene units, or one monoterpene or one sesquiterpene or diterpene group; R ⁴ and R ⁵ are hydrogen or lower alkyl; R ⁶ is lower alkyl; X is F or Cl or Br or I.

In the definitions of Chemical Formulas 1 and 2, the lower alkyl moiety of lower alkyl and lower alkanoyl is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl. It means a C1-C6 linear or branched alkyl group.

More specifically, the present invention relates to Asmarin A and Asmarin B extracted and isolated from the Lassilia species . The structure of the compound is as follows:

The stereochemical structure in the above formula shows the relative atomic arrangement.

Asmarin A and Asmarin B exhibit antitumor activity. In particular, Asmarin A and Asmarin B show antitumor activity against cell lines derived from solid tumors of humans such as human lung carcinoma, human colon carcinoma and human melanoma, and other tumor cells such as leukemias and lymphomas. It is also active against the line.

The present invention also provides a method of treating a mammal having a disease caused by a malignant tumor sensitive to said compound comprising administering a therapeutically effective amount of a compound having Formula 1 or Formula 2 or a pharmaceutical composition thereof.

The present invention also provides a pharmaceutical composition comprising as an active ingredient a compound having the formula (1) or (2), and providing a method for preparing the compound.

On the other hand, the present invention provides a method for preparing Asmarin A and Asmarin B comprising an extraction step and a separation step from Sponge raspilia species .

Examples of pharmaceutical compositions include solid phases (tablets, pills, capsules, granules, etc.) or liquids (solutions, suspensions, emulsions) having a suitable composition for oral, topical, or parenteral administration, and are pure compounds or carriers or other Mixtures of pharmaceutically active compounds. The composition of the present invention is sterilized when administered parenterally.

The exact dosage of a pharmaceutical composition comprising a compound having Formula 1 or Formula 2 will vary depending upon the formulation, mode of application, and the particular cactus, host and tumor to be treated. Other factors, such as age, weight, sex, food, time of administration, rate of excretion, the condition of the host, the composition of the drug, the sensitivity of the reaction, and the degree of disease should also be considered. Administration can be carried out continuously or periodically within the range of the maximum content.

Antitumor activity

Cells were maintained at the log stage of growth in Eagles minimal essential medium, which contained Earle's Balanced Salts, 2.0 mM L-glutamine, non-essential amino acids, but no sodium bicarbonate (EMEM / neaa), 10% fetal bovine serum (FCS), 10-2 M sodium bicarbonate and 0.1 g / l penicillin-G + streptomycin sulfate.

The screening procedure for determining and comparing the antitumor activity of the compounds of the present invention was carried out by applying the method described in Bergeron et al. (Reference 3). The antitumor cells used were P-388 (suspension culture of lymph neoplastic tumors of DBA / 2 mice), A-549 (monolayer culture of human lung carcinoma), HT-29 (monolayer of human colon carcinoma) Cultures) and MEL-28 (monolayer of human melanoma).

P-388 cells were seeded in 16 mm wells with 1 × 10 ⁴ cells per well in 1 ml aliquot of MEM 5FCS containing the indicated concentration of medicament. Separate sets of cultures without medicament were seeded as growth controls to check if the cells were in logarithmic growth phase. All decisions were made twice. Incubation at 37 ° C. under 10% CO ₂ atmosphere with 98% humidity for 3 days, followed by approximate IC ₅₀ by comparing growth in control wells with no drug addition and growth in wells with drug addition Was determined.

A-549, HT-29 and MEL-28 cells were seeded in 16 mm wells with 2 × 10 ⁴ cells per well in 1 ml aliquot of MEM 10FCS containing the indicated concentration of medication. Separate sets of cultures without medicament were seeded as growth controls to check if the cells were in logarithmic growth phase. All decisions were made twice. Incubation at 37 ° C. under 10% CO ₂ atmosphere with 98% humidity for 3 days and then the wells were stained with 0.1% crystal violet. Approximate IC ₅₀ was determined by comparing growth in control wells without drug addition with growth in wells with drug addition.

The results of in vitro cytotoxicity assays for Asmarin A and Asmarin B conducted using P-388, A-549, HT-29 and MEL-28 are shown in Table 1 below, expressed as IC ₅₀ (μM). Became:

P-388 A-549 HT-29 MEL-28 Asmarin A 1.18 1.18 1.18 1.18 Asmarin B 0.24 0.12 0.12 0.24

Extraction and separation

Low resolution mass spectra were measured with an EIMS mass spectrophotometer. ¹ H and ¹³ C-NMR spectra were measured with a Bruker ARX-500 spectrophotometer. All chemical shifts were measured relative to TMS (δ = 0 ppm).

The Lassilia species (Rob Van Soest), (river Demorspongia, thu poecilosclerida, and Raspailiidea) were collected in May 1997 by scuba diving at Dahlak Archipelago, Eritrea at 23.5 m. Standard samples are deposited with Tel Aviv University (ET-338). The collected sponges were frozen. The freeze-dried sponge (20 gr) was extracted with ethyl acetate 2x and evacuated to give 1.2 g of a brown gum. The brown gum was divided into four portions into Kupchon: hexane, carbon tetrachloride, chloroform and water. CHCl ₃ and CCl ₄ portions were similar. The two portions were combined, loaded onto a Sephadex LH-20 column and eluted with MeOH: CHCl 3 (1: 1) to give five splits. Partition (3-5) was repeatedly chromatographed again with a Sephadex LH-20 column and eluted using the same solvent to yield Asmarin A (100 mg), which was a solid, mp = 232 ° C., m / z ⁺ = 423 (C ₂₅ H ₃₇ N ₅ O), (100%) 188 (C ₈ H ₆ N ₅ O ⁺ ), [α] _D ²⁰ +55 ^o (c = 0.5, CHCl ₃ ), IR 3400 , 2928, 1600, 1553, 1451, 1400, 1388, 900 cm ^-1 , the structure of asmarin A was confirmed by X-ray analysis. Asmarin B (120 mg) was isolated using the same solvent, which was an oil, m / z ⁺ = 423 (C ₂₅ H ₃₇ N ₅ O), (100%) 188 (C ₈ H ₆ N ₅ O ⁺ ), [α] _D ²⁰ +60 ^o (c = 0.5, CHCl ₃ ), IR 3400, 2927, 1606, 1553, 1451, 1404, 1388, 900 cm ⁻¹ .

The NMR data of Asmarin A and Asmarin B are shown in Table 2: In all cases, the solvent is CDCl ₃ and all chemical shifts are for TMS (δ = 0 ppm).

δ _c δ _H C No. Asmarin A Asmarin B Asmarin A Asmarin B One 21.82 t 21.17 t 1.70 d, 1 H 1.45 m, 1 H 1.82 m, 2 H 2 28.56 t 24.13 t 1.85 brd, 1 H1.21 m, 1 H 1.75 m, 1 H1.60 m, 1 H 3 33.20 t 31.65 t 2.25 dt, 1H2.05 dd, 1H 2.45 m, 1 H 2.12 m, 1 H 4 160.59 s 153.65 s 5 40.05 s 39.35 s 6 37.24 t 38.12 t 1.50 m, 2 H 2.10 t, 1 H 1.20 t, 1 H 7 27.36 t 27.22 t 1.45 m, 2 H 1.54 m, 1H1.20 m, 1H 8 36.68 d 38.12 d 1.37 m, 1 H 1.35 m, 1 H 9 39.26 s 40.54 s 10 48.62 d 46.56 d 1.05 d, 1 H 1.39 m, 1 H 11 31.23 t 31.05 t 1.55 dt, 1 H1.25 m, 1 H 1.59 m, 1 H1.30 t, 1 H 12 33.01 t 31.55 t 1.95 dt, 1 H1.43 m, 1 H 1.95 dt, 1 H1.55 m, 1 H 13 64.20 s 64.95 s 14 36.68 t 36.40 t 2.50 dt, 1H 2.15 dd, 1H 2.55 m, 1H2.25 m, 1H 15 42.30 t 42.33 t 4.25 dt, 1 H 4.20 dd, 1 H 4.30 t, 2H 16 21.75 q 23.09 q 1.44 s, 3 H 1.49 s, 3H 17 15.93 q 15.84 q 0.70 d, 3H 0.74 d, 3 H 18 102.45 t 105.69 t 4.60 s, 2H 4.70 d, 2H 19 20.08 q 32.87 q 1.00 s, 3H 1.11 s, 3H 20 18.28 q 19.85 q 0.65 s, 3H 0.82 s, 3H 2' 151.68 d 151.64 d 8.50 s, 1 H 8.50 d, 1 H 4' 149.00 s 149.57 s 5 ' 109.31 s 109.32 s 6 ' 158.70 s 158.38 s 8' 143.10 d 143.32 d 7.95 s, 1 H 7.95 s, 1 H

Reference

1. Faulkner, D. Nat. Prod. Rep. 1997, 14, 259-302 and references therein

2. Nakamura, H. et al . , Tetrahedron Lett. 1984, 25, 2989-2922

3. Anti-tumor Activity of Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline, Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter, Spermidine Catecholamide Iron Chelate And anti-herpes activity, Biochem. Bioph. Res. Comm. 1984, 121, 848-854.

Claims (10)

The compound represented by the following formula Formula 1 Formula 2 In the above formula, R ¹ is hydrogen or lower alkyl or lower alkanoyl; R ² is hydrogen or lower alkyl; R ³ is alkyl comprising one or more isoprene units; R ⁴ and R ⁵ are hydrogen or lower alkyl; R ⁶ is lower alkyl; X is F or Cl or Br or I and the lower alkyl and lower alkyl groups of the lower alkanoyl are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and It means a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as hexyl. The compound of claim 1, wherein the compound is represented by the following Chemical Formula 3: Formula 3 The method of claim 1, wherein the compound is represented by the following formula (4) asmarin B: Formula 4 delete delete A pharmaceutical composition for treating malignant tumors comprising the compound having Formula 1 or Formula 2 of claim 1 as an active ingredient. A pharmaceutical composition for treating malignant tumors comprising the asmarin A of claim 2 or the asmarin B of claim 3 as an active ingredient. A method of treating malignant tumors in mammals other than humans, comprising administering a therapeutically effective amount of at least one other anti-tumor compound together with a compound of Formula 1 or Formula 2 as claimed in claim 1. A method of treating malignant tumors in mammals, except humans, comprising administering a therapeutically effective amount of at least one other anti-tumor compound together with Asmarin A of Claim 2 or Asmarin B of Claim 3. (. Raspailia sp) to (i) a sponge Ras file Ria species extracting with ethyl acetate; (ii) fractionating the extract of step (i) with hexane, carbon tetrachloride, chloroform and water; And (iii) eluting the fraction of step (ii) with MeOH: CHCl ₃ to produce asmarin A of claim 2 or asmarin B of claim 3.